The microorganisms that are mainly involved in the resistance process are the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) emphasizing their ability to escape from common antibacterial treatments. It has been significant public concern that the absence of progress in developing novel therapeutics to treat multidrug-resistant (MDR) infections caused by Gram-negative bacteria. We identified that a new pleuromutilin analog exhibited bactericidal activity against E. coli and Klebsiella pneumoniae with the MIC values of 0.78 and 0.65 g/mL, respectively. It is a very interesting fact that functionalizations of the carbonyl (C3-position) and the side chain (C14-position) of pleuromutilin can extend the spectrum of activity focused against Gram-negative bacteria. A pleuromutilin analog has been developed into a phase II clinical drug via systemic administration for the treatment of complicated skin and soft tissue infection (cSSTI) caused by Gram-positive pathogens, however, development of pleuromutilin analogs for Gram-negative bacterial infections has never been the subject of antibacterial researches. Our rationale for this application is that successful completion of the proposed projects will provide the critical date t perform extensive structure activity relationship studies of pleuromutilin derivatives for improvin PK/PD and in vivo efficacy. In order to accomplish the research projects proposed here, the PI has assembled an interdisciplinary consortium capable of fulfilling the goals of this proposal.